Methods for obtaining information from single cells within populations using DNA origami nanostructures without the need for single cell sorting

ABSTRACT

Methods for construction of DNA origami nanostructures, as well as for binding, isolation, linking, and deep sequencing information, such as both of TCR alpha and beta CDR3 mRNA, from individual cells within a mixed population of cells without the need for single cell sorting.

CROSS-REFERENCE

This application is a 371 application of PCT/US2014/041581 filed Jun. 9,2014, which claims priority to U.S. provisional patent application61/834,270 filed on Jun. 12, 2013, which are incorporated by referenceherein in their entirety.

FIELD OF INVENTION

This application relates to methods for obtaining genetic informationfrom individual cells within mixed cell populations without the need forsingle cell sorting. In some embodiments, methods are disclosed forconstruction of DNA origami nanostructures, for binding, isolation,linking, and deep sequencing of both TCR alpha and beta CDR3 mRNA fromindividual cells within a mixed population of cells.

BACKGROUND OF INVENTION

One cardinal property of the adaptive immune system is diversity: theimmune system must be able to recognize and respond to virtually anyinvading microorganism. In order to generate such diversity, developingB and T cells rearrange a defined set of variable (V), diversity (D),and joining (J) gene segments, with N-nucleotide addition andsubtraction at the joints of these gene segments, resulting in asemi-random CDR3 repertoire of immune receptors. Further diversity isgenerated by pairing of rearranged alpha and beta (for the T cellreceptor (TCR)) or heavy and light chain (for the B cell receptor(BCR)).

Current technologies allow for analysis of CDR3 diversity within eitherthe alpha or beta TCR (or heavy and light chain BCR), but no currentmethods exist for obtaining both CDR3 from individual cells from largepolyclonal populations: single cell sequencing remains too expensivewhile molecular strategies for obtaining linked CDR3 information fromsingle cells have not been adequately developed.

SUMMARY OF THE INVENTION

In certain embodiments, a methodology, including construction of DNAorigami nanostructures, for binding, isolation, linking, and deepsequencing of both TCR alpha and beta CDR3 mRNA from individual cellswithin a mixed population of cells is described. This represents aquantum advance in immunology, as no known methods are available forobtaining linked CDR3 information from individuals cells from largemixed populations of cells; current approaches are only able to obtainCDR3 sequence information on either the TCR alpha or TCR beta: suchstrategies employ lysis of mixed populations of cells resulting in“scrambling” of genomic DNA and mRNA for each TCR or BCR chain,precluding paired analysis.

DNA origami is the nanoscale folding of DNA to create arbitrary two andthree-dimensional shapes at the nanoscale. The specificity of theinteractions between complementary base pairs make DNA a usefulconstruction material, through design of its base sequences.

Exemplary DNA origami nanostructures are composed of ssDNA (M13 phage)refolded with complementary ssDNA “staple” sequences into computerdesign-aided predetermined shapes with selected staples extended withcomplementary sequences to TCR alpha and beta constant region mRNA.Methods for high efficiency transfection of primary T cells with thedeveloped structures, isolation of DNA origami from transfected cellswith specifically bound TCR mRNA, as well as a molecular approach forlinking the CDR3 from the TCR alpha and beta mRNA into a single cDNAmolecule for use in multiplex CDR3 paired end sequencing using existingtechnologies also are disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a DNA origami scaffold-staple layout for single layer DNAorigami objects using square lattice packing including fluorescein andTAMRA fret signals.

FIG. 2: The sequence of the short oligomer strands are generated usingTiamat software and are defined by the sequence of the scaffold and canbe extended to include a single-stranded “probe” sequences that extendfrom the DNA Origami structure. These sequences are complementary to theconserved regions of the TCR α or β mRNA coding sequences (pink and bluerespectively, or light and dark when reproduced in black and white),which have been estimated to maintain an “open” secondary structure asestablished by estimated RNA folding software. The location of thebiotin tags on the origami is displayed in black.

FIG. 3: Estimated “open” secondary structure areas of TCR β mRNA asestablished by predicting RNA folding software. The loops are areasdesignated for origami probe site attachment.

FIG. 4: Estimated “open” secondary structure areas of TCR α mRNA asestablished by predicting RNA folding software. The loops are areasdesignated for origami probe site attachment.

FIG. 5: Isolated nanostructures are visualized by atomic forcemicroscopy (AFM) to verify proper folding.

FIG. 6: Reverse transcription, T4 ligation and linkage of origami-boundmRNA to provide input for high throughput sequencing. Our first RTprimer attaches to the Cβ region, and utilizing the close proximity ofboth TCR chains maintained by our origami molecules, we employ a mix of19 additional 200 nt primers (each specific for a different Vβ gene)which act as a linker from Vβ to Cα. We then run a reverse transcriptionreaction with optimized temperature and RNase inhibitor concentrationthat reduces displacement activity of the RT enzyme. We then follow withan RNA-templated T4 DNA ligation step with optimized temperature, ATP,and enzyme concentrations to produce a TCRα/β hybrid cDNA molecule whichis then amplified by multiplex PCR using a single CP primer and amultiplex Vα primer mix (one for each Vα gene). The final product is apool of amplicons around 400 bp in length which serve as input materialfor high throughput sequencing.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Embodiments described herein relate to methods for high-efficiencytransfection and use of DNA origami nanostructures that are able to bindTCR alpha and beta mRNA within transfected cells, strategies forisolation of DNA origami with bound TCR mRNA, and a molecular approachfor linking both CDR3 into a single cDNA molecule for use in paired-enddeep sequencing. Thus, we have developed a novel strategy for obtaininglinked TCR CDR3 sequence information from single cells, without the needfor sorting individual cells, which can be used to analyze TCRrepertoires including diversity in the pre-immune repertoire as well assubpopulations of cells of interest.

Current approaches to obtaining linked information on CDR3 sequence fromindividual cells include single cell sorting followed by PCR andconventional sequencing, lysis of cells in oil emulsion droplets anddeep sequencing, and nucleic acid bridges. Single cell sorting remainstoo costly for analysis of large cell populations; each cell/reactioncurrently costs $1-$2 making analysis of T cell repertoires from even anindividual mouse (˜10^7 T cells) or human (˜10^12 T cells) unfeasible.Lysis of individual cells in oil emulsion droplets currently is onlyable to yield analysis of a maximum of 10^5 T cells from any givenindividual (or a maximum of 1% of the total TCR repertoire).Transfection of nucleic acid bridges into cells results in hybridstructures that are efficiently cleaved by nucleases within transfectedcells and destruction of the template, precluding analysis.

We have developed DNA origami nanostructures that are able to bind andprotect TCR mRNA within individual transfected T cells. A hurdle to suchapproaches is transfection efficiency: typically, primary T cellpopulations exhibit low transfection efficiency (between 10-15%). DNAorigami nanostructures have inherently high transfection efficiencyproperties resulting in >80% transfection efficiency after simpleelectroporation. Additionally, labeling the origami with a biotin tagand following cell lysis with streptavidin column purification allowsthe DNA origami nanostructures with bound cellular mRNA to bere-isolated from transfected cells with high efficiency and purity foruse in subsequent molecular reactions.

A final hurdle to obtaining linked information on TCR CDR3 sequencesfrom individual cells is that isolated mRNA species, bound to individualDNA origami nanostructures from individual cells, need to become linkedinto a single cDNA molecule for multiplex PCR, creating an ampliconsuitable for paired-end deep sequencing of the two CDR3 regions. We havedeveloped a molecular strategy, using a multi primer system with areverse transcription reaction that lacks substantial levels ofexonuclease activity (so as not to displace the downstream primer) andcommercially available T4 ligase to link the upstream and downstreamproducts, resulting in a single cDNA molecule with the TCRα CDR3 at oneend and the TCRβ CDR3 at the other. This can then be used with existingTCR multiplex V gene primers and a single Cβ primer to produce linkedinformation on both CDR3 regions in a single ˜400 bp DNA molecule forlarge populations of T cells which can then be used as input forillumina paired-end high throughput sequencing.

Currently, analysis of one TCR CDR3 is used as a diagnostic for disease(the immune response to an infection or tumor is diagnostic for the typeof infection or tumor). In addition, analysis of CDR3 sequences hasbecome a staple in both research applications to understand the immunesystem as well as in clinical applications for assessment of immunecompetency after immune reconstitution and during aging.

The methods disclosed herein are very adaptable. Essentially, the basictechnology used to create the DNA origami nanostructures could bemodified by changing the extended complementary staple sequences toallow for hybridization to any two mRNA species of interest for which itis important to understand the sequence of mRNA from individual cellswithin a mixed population of cells. For example, changing the identityof the probes to match the TCR gamma and delta constant regions, or tomatch IgH and Igl constant regions of B cell receptors, or to constantregions of immune receptors from other species (i.e. human), or to anytwo genes of interest.

Thus, while the following examples of the application of the methodsherein are given, they are for illustration only and not intended tolimit the claims.

DNA Origami Design: The design of the internal DNA origamiscaffold-staple layout for single layer DNA origami objects using squarelattice packing was accomplished with the software packages Tiamat [basestructure published by Rothemund, P. W. K. Folding DNA to createnanoscale shapes and patterns. Nature 440, 297-302 (2006)] (FIG. 1). Along circular single-stranded DNA derived from the bacteriophage M13mp18genome (Table 1 below; purchased from Affymetrix) is folded into athree-dimensional shape using 216 shorter ssDNA oligomer strands(sequences in Table 2 below; purchased from IDT) that direct folding ofthe longer M13mp18 ssDNA.

The sequences of the short oligomer strands are generated using Tiamatsoftware and are defined by the sequence of the scaffold. However, theycan be extended to include a single-stranded “probe” sequence thatextends from the DNA Origami structure (FIG. 2; sequences in Table 3below). These sequences are complementary to the conserved regions ofthe TCR α or β mRNA coding sequences which have been estimated tomaintain an “open” secondary structure as established by estimated RNAfolding software (FIG. 3). Site-directed attachment of fluorescent dyesTAMRA and FITC to staples 89 and 91 respectively can be included tofacilitate detection of transfected cells and subsequent isolation ofDNA origami nanostructures with bound TCR mRNA (FIGS. 1 and 2; Table 2).Biotinylation of staples 77, 78, 79 and 80 allows for monomeric avidinresin purification of DNA nanostructures after transfection and celllysis (FIG. 2; Table 2).

DNA Origami Refolding: The scaffold-staple layout specifies a structuralsolution for the mixture of scaffold DNA and staple molecules thatminimizes energy through Watson-Crick base-pairing. Single-strandedM13mp18 bacteriophage genome (7249 nt) is purchased from the commercialvendor Affymetrix. All oligonucleotide staples are synthesized andprocured from the commercial vendor IDT. Alpha and beta staple-probeoligonucleotides are purified and isolated by 10% denaturing-PAGE in1×TBE with 525 ul 10% APS and 29.4 ul TEMED and purified with CorningSpin X gel filter centrifuge tubes using a freeze-thaw cycle as follows.The PAGE gel is placed on a transluminator. A razor blade is used to cutout major bands from the denaturing-PAGE gel. Bands are chopped intosmall pieces and small gel blocks are collected into Corning Spin Xtubes. 500 uL elution buffer is then added to cut the gel. Samples arethen shaken overnight at RT (the aim is to loose the gel and let the DNAmigrate out from the pores of the gel into solution, this process isdiffusion limited, thus temperature dependent and takes time). Tubes arethen centrifuged 8000 rpm, 6 min to separate gel blocks from eluted DNA.1000 uL butyl alcohol is then added and the tubes are vortexed for 1min, centrifuged at 2000 rpm, 1 min. The upper layer of butyl alcohol isremoved by pipetting (this step is to extract any organic soluble fromthe DNA sample i.e. EB and tracking dyes). 1000 uL 70% ethyl alcohol isthen added and mixed well. Samples are then incubated at −20 C, 2 hr toprecipitate DNA. Samples are then centrifuged at 13000 rpm, 30 min at 4C to pellet the DNA (DNA is not soluble in 70% ethanol). The ethanol isthen discarded. Samples are then dried by vacufuging for 2 hr at 30 C.50 uL nanopure H₂O is then added, samples are vortexed for 1 min todissolve purified DNA fragments.

Staple oligonucleotides are then standardized to 30 pmol/ul by measuringlight absorbance at 260 nm then mixed in equamolar amounts resulting ina master pool with each staple present at 500 nM. Scaffold M13mp18 ssDNAand staple DNA are mixed at a fixed 5:1 stoichiometric ratio (20 nMscaffold, 100 nM each staple) in pH-stabilizing 1×TAE-MG2+ aqueousbuffer, followed by thermal denaturation (80° C.) and annealing (23° C.)for 4 hours.

DNA Origami Analysis: Folded DNA origami species are purified fromnon-folded products and unused primers by washing with butyl alcoholfollowed by isopropanol, and elution in 50 uL nanopure water andcentrifugation through 100K nominal molecular weight limit (NMWL) Amiconmicrocolumn filters. Purification typically results in a solutioncontaining 2-5 nM of the target DNA origami nanostructure. DNA origamiconcentration is measured by A260/A280 absorbance and standardization to50 nM. Isolated nanostructures are visualized by atomic force microscopy(AFM) to verify proper folding (FIG. 4).

Transfection of DNA origami into T cells: Splenocytes from 4-6 week oldC57BL/6 mice are prepared by mechanical disruption and red blood celllysis (0.83% NH4Cl). CD8 T cells are then purified by magnetic cellsorting (MACS Miltenyi Biotech) and >95% purity of sorted populationsconfirmed by flow cytometry. Cells are pelleted by centrifugation (1200rpm, 5 min, 4 C), washed with OPTI-MEM media (Invitrogen), andresuspended in OPTI-MEM media at 5×10^6 cells/ml. For electroporation,the ECM 830 Square Wave Electroporation System (Harvard Apparatus BTX,Holliston, Mass., USA) is used with the cuvette safety stand attachmentand 2.0 mm gap cuvettes (Harvard Apparatus, BTX) using the followingsettings: Mode=LV, 300 V, 5 ms, 1 pulse, 1.5 kV/cm desired fieldstrength. Samples consist of 100 uL (5×10^6 cells/ml) cell suspensionand 25 uL (50 nM) DNA origami suspension in 1×TAE-Mg²⁺. Immediatelyafter electroporation, cells are transferred to a 96 well plate,cuvettes are rinsed with 100 uL fresh culture RPMI-1640 medium with 10%fetal calf serum which is added to the sample and the plates areincubated at 37 C for 24 h. To assess transfection efficiency, cells arestained with anti-CD8-APC antibody (1:100 dilution, BD Biosciences) andimmediately acquired on a LSR Fortessa flow cytometer. The DNA origamicontain a fluorescein isothiocyanate (FITC; 488 nm excitation, 518 nmemission) tag, and successfully transfected CD8 T cells can beidentified by FACS.

Reisolation and purification of origami with bound mRNA: Transfectedcells are pelleted by centrifugation at 1300 rpm for 3 min, thesupernatant is decanted and the cells are then lysed with 100 uL 1%NP-40 lysis buffer (Thermo Scientific) for 1 hr on ice. Origami fromtransfected cells are purified by subjecting transfected cell lysate tostreptavidin column filtration (Thermo Scientific Streptavidin AgaroseResin; Sigma Prep Column, 500 uL, 7-20 um pore size). 50 uL resin isadded to the Prep column, the column is then centrifuged at 2000 rpm for10 s to remove the storage buffer. The resin is washed with 500 uL1×TAE^(Mg2+) and centrifuged at 2000 rpm for 10 s. The column is cappedand the cell lysate (containing the biotinylated DNA sample) is thenincubated with resin in the column for 30 min at RT, shaking by handevery 10 min. The column is then uncapped and the unbound mRNA andcellular debris is washed away using 500 uL 1×TAE^(Mg2+) and centrifugedat 2000 rpm for 10 s, five times. The column is then recapped beforereverse transcription.

Reverse transcription and linkage of bound mRNA to provide input forhigh throughput sequencing: After reisolation and purification oforigami with bound mRNA, a dual-primer linkage reverse transcriptionreaction followed by a T4 ligation reaction is performed directly in thepurification column to produce cDNA molecules which can then bemultiplex-PCR amplified to provide input material for Illumina pairedend high throughput sequencing. The first RT primer attaches to an openarea of the C13 region, and utilizing the close proximity (andmeasurable distance) of both TCR chains maintained by our origamimolecules, the second set of primers consist of a multiplex pool whereeach primer is 5′-phosphorylated and acts as a linker from one specificVβ to Cα (FIG. 6; Table 4). We then run a reverse transcription reactionusing the Omniscript RT kit (Qiagen) for 60 min, 37 C, supplemented with1 uL RiboLock RNase Inhibitor (Thermo Scientific, 40 U/uL), whichresults in reduced displacement activity of the RT enzyme. We thenfollow with an RNA-templated T4 DNA ligation step with optimizedtemperature, duration and ATP concentration to produce a TCRα/β hybridcDNA molecule (FIG. 6). After ligation, the column is heated to 95 C for5 min to degrade the origami and dissociate the cDNA from the mRNA. Thecolumn is then centrifuged at 2000 rpm for 30 s to elute the cDNA foruse in the following multiplex PCR reaction.

Reverse transcription reactions (Omniscript, Qiagen) are performed underconditions that maximize primer annealing and minimize stranddisplacement activity of the reverse transcriptase enzyme: 15 uL diH₂O,2 uL Omniscript buffer, 2 uL dNTPs (5 mM each), 1 uL RiboLock RNaseinhibitor (Thermo Scientific), 1 uL constant alpha primer (100 μM)(Table 4), 3 uL (10-20 uM each) variable beta multiplex linker primersolution (Table 4) and 1 uL reverse transcriptase enzyme is prepared ina PCR tube and added directly to the capped sample purification columnand incubated at 37 C for 60 min in a heat block.

RNA-templated T4 DNA ligation is then performed: 7 uL T4 DNA ligasebuffer (New England Biolabs) and 2 uL T4 DNA ligase (New EnglandBiolabs) is prepared in a PCR tube and added directly to the cappedsample purification columns. The reactions are incubated at RT for 60min. The caps are removed from the columns and the enzymes are heatinactivated a long with dissociation of origami and mRNA from theligated cDNA by incubating the columns in a 95 C heat block for 5 min.The ligated cDNA is then eluted from the column by centrifuging at 2000rpm for 30 s. Collected cDNA is kept on ice until use in following PCRreaction.

Multiplex PCR amplification of TCRα/β CDR3 cDNA hybrid molecules:Standard multiplex PCR is performed on the cDNA molecules produced afterreverse transcription and T4 ligation reactions utilizing a single 5′phosphorylated Cβ primer (Table 5) and a multiplex 5′ phosphorylated Vαprimer solution (Table 5). This use of a Taq polymerase results in finalDNA molecules (amplicons) consisting of 400-500 bp (FIG. 5) spanningCα(20 bp)-Jα(variable)-Vα(20 bp)-Linker(200 bp)-Cβ(20bp)-Jβ(variable)-Dβ(variable)-Vβ(20 bp) (FIG. 5) with 3′A overhangs.Each amplicon is also 5′phosphorylated due to the attached 5′Phos oneach primer, allowing for simple ligation of Illumina specificsequencing adaptors per manufacturer's protocol.

TABLE 1 M13mp18 Phage DNA Sequence:AATGCTACTACTATTAGTAGAATTGATGCCACCTTTTCAGCTCGCGCCCCAAATGAAAATATAGCTAAACAGGTTATTGACCATTTGCGAAATGTATCTAATGGTCAAACTAAATCTACTCGTTCGCAGAATTGGGAATCAACTGTTATATGGAATGAAACTTCCAGACACCGTACTTTAGTTGCATATTTAAAACATGTTGAGCTACAGCATTATATTCAGCAATTAAGCTCTAAGCCATCCGCAAAAATGACCTCTTATCAAAAGGAGCAATTAAAGGTACTCTCTAATCCTGACCTGTTGGAGTTTGCTTCCGGTCTGGTTCGCTTTGAAGCTCGAATTAAAACGCGATATTTGAAGTCTTTCGGGCTTCCTCTTAATCTTTTTGATGCAATCCGCTTTGCTTCTGACTATAATAGTCAGGGTAAAGACCTGATTTTTGATTTATGGTCATTCTCGTTTTCTGAACTGTTTAAAGCATTTGAGGGGGATTCAATGAATATTTATGACGATTCCGCAGTATTGGACGCTATCCAGTCTAAACATTTTACTATTACCCCCTCTGGCAAAACTTCTTTTGCAAAAGCCTCTCGCTATTTTGGTTTTTATCGTCGTCTGGTAAACGAGGGTTATGATAGTGTTGCTCTTACTATGCCTCGTAATTCCTTTTGGCGTTATGTATCTGCATTAGTTGAATGTGGTATTCCTAAATCTCAACTGATGAATCTTTCTACCTGTAATAATGTTGTTCCGTTAGTTCGTTTTATTAACGTAGATTTTTCTTCCCAACGTCCTGACTGGTATAATGAGCCAGTTCTTAAAATCGCATAAGGTAATTCACAATGATTAAAGTTGAAATTAAACCATCTCAAGCCCAATTTACTACTCGTTCTGGTGTTTCTCGTCAGGGCAAGCCTTATTCACTGAATGAGCAGCTTTGTTACGTTGATTTGGGTAATGAATATCCGGTTCTTGTCAAGATTACTCTTGATGAAGGTCAGCCAGCCTATGCGCCTGGTCTGTACACCGTTCATCTGTCCTCTTTCAAAGTTGGTCAGTTCGGTTCCCTTATGATTGACCGTCTGCGCCTCGTTCCGGCTAAGTAACATGGAGCAGGTCGCGGATTTCGACACAATTTATCAGGCGATGATACAAATCTCCGTTGTACTTTGTTTCGCGCTTGGTATAATCGCTGGGGGTCAAAGATGAGTGTTTTAGTGTATTCTTTTGCCTCTTTCGTTTTAGGTTGGTGCCTTCGTAGTGGCATTACGTATTTTACCCGTTTAATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAGCCGTTGCTACCCTCGTTCCGATGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCAAAAGCGGCCTTTAACTCCCTGCAAGCCTCAGCGACCGAATATATCGGTTATGCGTGGGCGATGGTTGTTGTCATTGTCGGCGCAACTATCGGTATCAAGCTGTTTAAGAAATTCACCTCGAAAGCAAGCTGATAAACCGATACAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCTTTAGTTGTTCCTTTCTATTCTCACTCCGCTGAAACTGTTGAAAGTTGTTTAGCAAAATCCCATACAGAAAATTCATTTACTAACGTCTGGAAAGACGACAAAACTTTAGATCGTTACGCTAACTATGAGGGCTGTCTGTGGAATGCTACAGGCGTTGTAGTTTGTACTGGTGACGAAACTCAGTGTTACGGTACATGGGTTCCTATTGGGCTTGCTATCCCTGAAAATGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTTCTGAGGGTGGCGGTACTAAACCTCCTGAGTACGGTGATACACCTATTCCGGGCTATACTTATATCAACCCTCTCGACGGCACTTATCCGCCTGGTACTGAGCAAAACCCCGCTAATCCTAATCCTTCTCTTGAGGAGTCTCAGCCTCTTAATACTTTCATGTTTCAGAATAATAGGTTCCGAAATAGGCAGGGGGCATTAACTGTTTATACGGGCACTGTTACTCAAGGCACTGACCCCGTTAAAACTTATTACCAGTACACTCCTGTATCATCAAAAGCCATGTATGACGCTTACTGGAACGGTAAATTCAGAGACTGCGCTTTCCATTCTGGCTTTAATGAGGATTTATTTGTTTGTGAATATCAAGGCCAATCGTCTGACCTGCCTCAACCTCCTGTCAATGCTGGCGGCGGCTCTGGTGGTGGTTCTGGTGGCGGCTCTGAGGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGGGAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAAGATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAACGCGCTACAGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGATTACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCCCAAATGGCTCAAGTCGGTGACGGTGATAATTCACCTTTAATGAATAATTTCCGTCAATATTTACCTTCCCTCCCTCAATCGGTTGAATGTCGCCCTTTTGTCTTTGGCGCTGGTAAACCATATGAATTTTCTATTGATTGTGACAAAATAAACTTATTCCGTGGTGTCTTTGCGTTTCTTTTATATGTTGCCACCTTTATGTATGTATTTTCTACGTTTGCTAACATACTGCGTAATAAGGAGTCTTAATCATGCCAGTTCTTTTGGGTATTCCGTTATTATTGCGTTTCCTCGGTTTCCTTCTGGTAACTTTGTTCGGCTATCTGCTTACTTTTCTTAAAAAGGGCTTCGGTAAGATAGCTATTGCTATTTCATTGTTTCTTGCTCTTATTATTGGGCTTAACTCAATTCTTGTGGGTTATCTCTCTGATATTAGCGCTCAATTACCCTCTGACTTTGTTCAGGGTGTTCAGTTAATTCTCCCGTCTAATGCGCTTCCCTGTTTTTATGTTATTCTCTCTGTAAAGGCTGCTATTTTCATTTTTGACGTTAAACAAAAAATCGTTTCTTATTTGGATTGGGATAAATAATATGGCTGTTTATTTTGTAACTGGCAAATTAGGCTCTGGAAAGACGCTCGTTAGCGTTGGTAAGATTCAGGATAAAATTGTAGCTGGGTGCAAAATAGCAACTAATCTTGATTTAAGGCTTCAAAACCTCCCGCAAGTCGGGAGGTTCGCTAAAACGCCTCGCGTTCTTAGAATACCGGATAAGCCTTCTATATCTGATTTGCTTGCTATTGGGCGCGGTAATGATTCCTACGATGAAAATAAAAACGGCTTGCTTGTTCTCGATGAGTGCGGTACTTGGTTTAATACCCGTTCTTGGAATGATAAGGAAAGACAGCCGATTATTGATTGGTTTCTACATGCTCGTAAATTAGGATGGGATATTATTTTTCTTGTTCAGGACTTATCTATTGTTGATAAACAGGCGCGTTCTGCATTAGCTGAACATGTTGTTTATTGTCGTCGTCTGGACAGAATTACTTTACCTTTTGTCGGTACTTTATATTCTCTTATTACTGGCTCGAAAATGCCTCTGCCTAAATTACATGTTGGCGTTGTTAAATATGGCGATTCTCAATTAAGCCCTACTGTTGAGCGTTGGCTTTATACTGGTAAGAATTTGTATAACGCATATGATACTAAACAGGCTTTTTCTAGTAATTATGATTCCGGTGTTTATTCTTATTTAACGCCTTATTTATCACACGGTCGGTATTTCAAACCATTAAATTTAGGTCAGAAGATGAAATTAACTAAAATATATTTGAAAAAGTTTTCTCGCGTTCTTTGTCTTGCGATTGGATTTGCATCAGCATTTACATATAGTTATATAACCCAACCTAAGCCGGAGGTTAAAAAGGTAGTCTCTCAGACCTATGATTTTGATAAATTCACTATTGACTCTTCTCAGCGTCTTAATCTAAGCTATCGCTATGTTTTCAAGGATTCTAAGGGAAAATTAATTAATAGCGACGATTTACAGAAGCAAGGTTATTCACTCACATATATTGATTTATGTACTGTTTCCATTAAAAAAGGTAATTCAAATGAAATTGTTAAATGTAATTAATTTTGTTTTCTTGATGTTTGTTTCATCATCTTCTTTTGCTCAGGTAATTGAAATGAATAATTCGCCTCTGCGCGATTTTGTAACTTGGTATTCAAAGCAATCAGGCGAATCCGTTATTGTTTCTCCCGATGTAAAAGGTACTGTTACTGTATATTCATCTGACGTTAAACCTGAAAATCTACGCAATTTCTTTATTTCTGTTTTACGTGCAAATAATTTTGATATGGTAGGTTCTAACCCTTCCATTATTCAGAAGTATAATCCAAACAATCAGGATTATATTGATGAATTGCCATCATCTGATAATCAGGAATATGATGATAATTCCGCTCCTTCTGGTGGTTTCTTTGTTCCGCAAAATGATAATGTTACTCAAACTTTTAAAATTAATAACGTTCGGGCAAAGGATTTAATACGAGTTGTCGAATTGTTTGTAAAGTCTAATACTTCTAAATCCTCAAATGTATTATCTATTGACGGCTCTAATCTATTAGTTGTTAGTGCTCCTAAAGATATTTTAGATAACCTTCCTCAATTCCTTTCAACTGTTGATTTGCCAACTGACCAGATATTGATTGAGGGTTTGATATTTGAGGTTCAGCAAGGTGATGCTTTAGATTTTTCATTTGCTGCTGGCTCTCAGCGTGGCACTGTTGCAGGCGGTGTTAATACTGACCGCCTCACCTCTGTTTTATCTTCTGCTGGTGGTTCGTTCGGTATTTTTAATGGCGATGTTTTAGGGCTATCAGTTCGCGCATTAAAGACTAATAGCCATTCAAAAATATTGTCTGTGCCACGTATTCTTACGCTTTCAGGTCAGAAGGGTTCTATCTCTGTTGGCCAGAATGTCCCTTTTATTACTGGTCGTGTGACTGGTGAATCTGCCAATGTAAATAATCCATTTCAGACGATTGAGCGTCAAAATGTAGGTATTTCCATGAGCGTTTTTCCTGTTGCAATGGCTGGCGGTAATATTGTTCTGGATATTACCAGCAAGGCCGATAGTTTGAGTTCTTCTACTCAGGCAAGTGATGTTATTACTAATCAAAGAAGTATTGCTACAACGGTTAATTTGCGTGATGGACAGACTCTTTTACTCGGTGGCCTCACTGATTATAAAAACACTTCTCAGGATTCTGGCGTACCGTTCCTGTCTAAAATCCCTTTAATCGGCCTCCTGTTTAGCTCCCGCTCTGATTCTAACGAGGAAAGCACGTTATACGTGCTCGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGAACCACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCTTTGCCTGGTTTCCGGCACCAGAAGCGGTGCCGGAAAGCTGGCTGGAGTGCGATCTTCCTGAGGCCGATACTGTCGTCGTCCCCTCAAACTGGCAGATGCACGGTTACGATGCGCCCATCTACACCAACGTGACCTATCCCATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAATGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGCGAATTATTTTTGATGGCGTTCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAATGCGAATTTTAACAAAATATTAACGTTTACAATTTAAATATTTGCTTATACAATCTTCCTGTTTTTGGGGCTTTTCTGATTATCAACCGGGGTACATATGATTGACATGCTAGTTTTACGATTACCGTTCATCGATTCTCTTGTTTGCTCCAGACTCTCAGGCAATGACCTGATAGCCTTTGTAGATCTCTCAAAAATAGCTACCCTCTCCGGCATTAATTTATCAGCTAGAACGGTTGAATATCATATTGATGGTGATTTGACTGTCTCCGGCCTTTCTCACCCTTTTGAATCTTTACCTACACATTACTCAGGCATTGCATTTAAAATATATGAGGGTTCTAAAAATTTTTATCCTTGCGTTGAAATAAAGGCTTCTCCCGCAAAAGTATTACAGGGTCATAATGTTTTTGGTACAACCGATTTAGCTTTATGCTCTGAGGCTTTATTGCTTAATTTTGCTAATTCTTTGCCTTGCCTGTATGATTTATTGGATGTT (SEQ ID NO. 1)

TABLE 2 Staple sequences of DNA origami: Name Sequence   1CAAGCCCAATAGGAAC CCATGTACAAACAGTT (SEQ ID NO.2)   2AATGCCCCGTAACAGT GCCCGTATCTCCCTCA (SEQ ID NO. 3)   3TGCCTTGACTGCCTAT TTCGGAACAGGGATAG (SEQ ID NO. 4)   4GAGCCGCCCCACCACC GGAACCGCGACGGAAA (SEQ ID NO. 5)   5AACCAGAGACCCTCAG AACCGCCAGGGGTCAG (SEQ ID NO. 6)   6TTATTCATAGGGAAGG TAAATATT CATTCAGT (SEQ ID NO. 7)   7CATAACCCGAGGCATA GTAAGAGC TTTTTAAG (SEQ ID NO. 8)   8ATTGAGGGTAAAGGTG AATTATCAATCACCGG (SEQ ID NO. 9)   9AAAAGTAATATCTTAC CGAAGCCCTTCCAGAG (SEQ ID NO. 10)  10GCAATAGCGCAGATAG CCGAACAATTCAACCG (SEQ ID NO. 11)  11CCTAATTTACGCTAAC GAGCGTCTAATCAATA (SEQ ID NO. 12)  12TCTTACCAGCCAGTTA CAAAATAAATGAAATA (SEQ ID NO. 13)  13ATCGGCTGCGAGCATG TAGAAACCTATCATAT (SEQ ID NO. 14)  14CTAATTTATCTTTCCT TATCATTCATCCTGAA (SEQ ID NO. 15)  15GCGTTATAGAAAAAGC CTGTTTAG AAGGCCGG (SEQ ID NO. 16)  16GCTCATTTTCGCATTA AATTTTTG AGCTTAGA (SEQ ID NO. 17)  17AATTACTACAAATTCT TACCAGTAATCCCATC (SEQ ID NO. 18)  18TTAAGACGTTGAAAAC ATAGCGATAACAGTAC (SEQ ID NO. 19)  19TAGAATCCCTGAGAAG AGTCAATAGGAATCAT (SEQ ID NO. 20)  20CTTTTACACAGATGAA TATACAGTAAACAATT (SEQ ID NO. 21)  21TTTAACGTTCGGGAGA AACAATAATTTTCCCT (SEQ ID NO. 22)  22CGACAACTAAGTATTA GACTTTACAATACCGA (SEQ ID NO. 23)  23GGATTTAGCGTATTAA ATCCTTTGTTTTCAGG (SEQ ID NO. 24)  24ACGAACCAAAACATCG CCATTAAA TGGTGGTT (SEQ ID NO. 25)  25GAACGTGGCGAGAAAG GAAGGGAA CAAACTAT (SEQ ID NO. 26)  26TAGCCCTACCAGCAGA AGATAAAAACATTTGA (SEQ ID NO. 27)  27CGGCCTTGCTGGTAAT ATCCAGAACGAACTGA (SEQ ID NO. 28)  28CTCAGAGCCACCACCC TCATTTTCCTATTATT(SEQ ID NO. 29)  29CTGAAACAGGTAATAA GTTTTAACCCCTCAGA (SEQ ID NO. 30)  30AGTGTACTTGAAAGTA TTAAGAGGCCGCCACC (SEQ ID NO. 31)  31GCCACCACTCTTTTCA TAATCAAACCGTCACC (SEQ ID NO. 32)  32GTTTGCCACCTCAGAG CCGCCACCGATACAGG (SEQ ID NO. 33)  33GACTTGAGAGACAAAA GGGCGACAAGTTACCA (SEQ ID NO. 34)  34AGCGCCAACCATTTGG GAATTAGATTATTAGC (SEQ ID NO. 35)  35GAAGGAAAATAAGAGC AAGAAACAACAGCCAT (SEQ ID NO. 36)  36GCCCAATACCGAGGAA ACGCAATAGGTTTACC (SEQ ID NO. 37)  37ATTATTTAACCCAGCT ACAATTTTCAAGAACG (SEQ ID NO. 38)  38TATTTTGCTCCCAATC CAAATAAGTGAGTTAA (SEQ ID NO. 39)  39GGTATTAAGAACAAGA AAAATAATTAAAGCCA (SEQ ID NO. 40)  40TAAGTCCTACCAAGTA CCGCACTCTTAGTTGC (SEQ ID NO. 41)  41ACGCTCAAAATAAGAA TAAACACCGTGAATTT (SEQ ID NO. 42)  42AGGCGTTACAGTAGGG CTTAATTGACAATAGA (SEQ ID NO. 43)  43ATCAAAATCGTCGCTA TTAATTAACGGATTCG (SEQ ID NO. 44)  44CTGTAAATCATAGGTC TGAGAGACGATAAATA (SEQ ID NO. 45)  45CCTGATTGAAAGAAAT TGCGTAGACCCGAACG (SEQ ID NO. 46)  46ACAGAAATCTTTGAAT ACCAAGTTCCTTGCTT (SEQ ID NO. 47)  47TTATTAATGCCGTCAA TAGATAATCAGAGGTG (SEQ ID NO. 48)  48AGATTAGATTTAAAAG TTTGAGTACACGTAAA (SEQ ID NO. 49)  49AGGCGGTCATTAGTCT TTAATGCGCAATATTA (SEQ ID NO. 50)  50GAATGGCTAGTATTAA CACCGCCTCAACTAAT (SEQ ID NO. 51)  51CCGCCAGCCATTGCAA CAGGAAAAATATTTTT (SEQ ID NO. 52)  52CCCTCAGAACCGCCAC CCTCAGAACTGAGACT (SEQ ID NO. 53)  53CCTCAAGAATACATGG CTTTTGATAGAACCAC (SEQ ID NO. 54)  54TAAGCGTCGAAGGATT AGGATTAGTACCGCCA (SEQ ID NO. 55)  55CACCAGAGTTCGGTCA TAGCCCCCGCCAGCAA (SEQ ID NO. 56)  56TCGGCATTCCGCCGCC AGCATTGACGTTCCAG (SEQ ID NO. 57)  57AATCACCAAATAGAAA ATTCATATATAACGGA (SEQ ID NO. 58)  58TCACAATCGTAGCACC ATTACCATCGTTTTCA (SEQ ID NO. 59)  59ATACCCAAGATAACCC ACAAGAATAAACGATT (SEQ ID NO. 60)  60ATCAGAGAAAGAACTG GCATGATTTTATTTTG (SEQ ID NO. 61)  61TTTTGTTTAAGCCTTA AATCAAGAATCGAGAA (SEQ ID NO. 62)  62AGGTTTTGAACGTCAA AAATGAAAGCGCTAAT (SEQ ID NO. 63)  63CAAGCAAGACGCGCCT GTTTATCAAGAATCGC (SEQ ID NO. 64)  64AATGCAGACCGTTTTT ATTTTCATCTTGCGGG (SEQ ID NO. 65)  65CATATTTAGAAATACC GACCGTGTTACCTTTT (SEQ ID NO. 66)  66AATGGTTTACAACGCC AACATGTAGTTCAGCT (SEQ ID NO. 67)  67TAACCTCCATATGTGA GTGAATAAACAAAATC (SEQ ID NO. 68)  68AAATCAATGGCTTAGG TTGGGTTACTAAATTT (SEQ ID NO. 69)  69GCGCAGAGATATCAAA ATTATTTGACATTATC (SEQ ID NO. 70)  70AACCTACCGCGAATTA TTCATTTCCAGTACAT (SEQ ID NO. 71)  71ATTTTGCGTCTTTAGG AGCACTAAGCAACAGT (SEQ ID NO. 72)  72CTAAAATAGAACAAAG AAACCACCAGGGTTAG (SEQ ID NO. 73)  73GCCACGCTATACGTGG CACAGACAACGCTCAT (SEQ ID NO. 74)  74GCGTAAGAGAGAGCCA GCAGCAAAAAGGTTAT (SEQ ID NO. 75)  75GGAAATACCTACATTT TGACGCTCACCTGAAA (SEQ ID NO. 76)  76TATCACCGTACTCAGG AGGTTTAGCGGGGTTT (SEQ ID NO. 77)  77TGCTCAGTCAGTCTCT GAATTTACCAGGAGGT (SEQ ID NO. 78)  78GGAAAGCGACCAGGCG GATAAGTGAATAGGTG (SEQ ID NO. 79)  79TGAGGCAGGCGTCAGA CTGTAGCGTAGCAAGG (SEQ ID NO. 80)  80TGCCTTTAGTCAGACG ATTGGCCTGCCAGAAT (SEQ ID NO. 81)  81CCGGAAACACACCACG GAATAAGTAAGACTCC (SEQ ID NO. 82)  82ACGCAAAGGTCACCAA TGAAACCAATCAAGTT (SEQ ID NO. 83)  83TTATTACGGTCAGAGG GTAATTGAATAGCAGC (SEQ ID NO. 84)  84TGAACAAACAGTATGT TAGCAAACTAAAAGAA (SEQ ID NO. 85)  85CTTTACAGTTAGCGAA CCTCCCGACGTAGGAA (SEQ ID NO. 86)  86GAGGCGTTAGAGAATA ACATAAAAGAACACCC (SEQ ID NO. 87)  87TCATTACCCGACAATA AACAACATATTTAGGC (SEQ ID NO. 88)  88CCAGACGAGCGCCCAA TAGCAAGCAAGAACGC (SEQ ID NO. 89)  89AGAGGCATAATTTCAT CTTCTGACTATAACTA (SEQ ID NO. 90)  90TTTTAGTTTTTCGAGC CAGTAATAAATTCTGT (SEQ ID NO. 91)  91TATGTAAACCTTTTTT AATGGAAAAATTACCT (SEQ ID NO. 92)  92TTGAATTATGCTGATG CAAATCCACAAATATA (SEQ ID NO. 93)  93GAGCAAAAACTTCTGA ATAATGGAAGAAGGAG (SEQ ID NO. 94)  94TGGATTATGAAGATGA TGAAACAAAATTTCAT (SEQ ID NO. 95)  95CGGAATTATTGAAAGG AATTGAGGTGAAAAAT (SEQ ID NO. 96)  96ATCAACAGTCATCATA TTCCTGATTGATTGTT (SEQ ID NO. 97)  97CTAAAGCAAGATAGAA CCCTTCTGAATCGTCT (SEQ ID NO. 98)  98GCCAACAGTCACCTTG CTGAACCTGTTGGCAA (SEQ ID NO. 99)  99GAAATGGATTATTTAC ATTGGCAGACATTCTG (SEQ ID NO. 100) 100TTTT TATAAGTA TAGCCCGGCCGTCGAG (SEQ ID NO. 101) 101AGGGTTGA TTTT ATAAATCC TCATTAAATGATATTC (SEQ ID NO. 102) 102ACAAACAA TTTT AATCAGTA GCGACAGATCGATAGC (SEQ ID NO. 103) 103AGCACCGT TTTT TAAAGGTG GCAACATAGTAGAAAA (SEQ ID NO. 104) 104TACATACA TTTT GACGGGAG AATTAACTACAGGGAA (SEQ ID NO. 105) 105GCGCATTA TTTT GCTTATCC GGTATTCTAAATCAGA (SEQ ID NO. 106) 106TATAGAAG TTTT CGACAAAA GGTAAAGTAGAGAATA (SEQ ID NO. 107) 107TAAAGTAC TTTT CGCGAGAA AACTTTTTATCGCAAG (SEQ ID NO. 108) 108ACAAACAA TTTT ATTAATTA CATTTAACACATCAAG (SEQ ID NO. 109) 109AAAACAAA TTTT TTCATCAA TATAATCCTATCAGAT(SEQ ID NO. 110) 110GATGGCAA TTTT AATCAATA TCTGGTCACAAATATC (SEQ ID NO. 111) 111AAACCCTC TTTT ACCAGTAA TAAAAGGGATTCACCA GTCACACG TTTT (SEQ ID NO. 112)112 CCGAAATCCGAAAATC CTGTTTGAAGCCGGAA (SEQ ID NO. 113) 113CCAGCAGGGGCAAAATCCCTTATAAAGCCGGC (SEQ ID NO. 114) 114GCATAAAGTTCCACAC AACATACGAAGCGCCA (SEQ ID NO. 115) 115GCTCACAATGTAAAGCCTGGGGTGGGTTTGCC (SEQ ID NO. 116) 116TTCGCCATTGCCGGAA ACCAGGCATTAAATCA (SEQ ID NO. 117) 117GCTTCTGGTCAGGCTGCGCAACTGTGTTATCC (SEQ ID NO. 118) 118GTTAAAATTTTAACCAATAGGAACCCGGCACC (SEQ ID NO. 119) 119AGACAGTCATTCAAAA GGGTGAGAAGCTATAT (SEQ ID NO. 120) 120AGGTAAAGAAATCACCATCAATATAATATTTT (SEQ ID NO. 121) 121TTTCATTTGGTCAATA ACCTGTTTATATCGCG (SEQ ID NO. 122) 122TCGCAAATGGGGCGCGAGCTGAAATAATGTGT (SEQ ID NO. 123) 123TTTTAATTGCCCGAAA GACTTCAAAACACTAT (SEQ ID NO. 124) 124AAGAGGAACGAGCTTCAAAGCGAAGATACATT (SEQ ID NO. 125) 125GGAATTACTCGTTTACCAGACGACAAAAGATT (SEQ ID NO. 126) 126GAATAAGGACGTAACA AAGCTGCTCTAAAACA (SEQ ID NO. 127) 127CCAAATCACTTGCCCTGACGAGAACGCCAAAA (SEQ ID NO. 128) 128CTCATCTTGAGGCAAA AGAATACAGTGAATTT (SEQ ID NO. 129) 129AAACGAAATGACCCCCAGCGATTATTCATTAC (SEQ ID NO. 130) 130CTTAAACATCAGCTTG CTTTCGAGCGTAACAC (SEQ ID NO. 131) 131TCGGTTTAGCTTGATACCGATAGTCCAACCTA (SEQ ID NO. 132) 132TGAGTTTCGTCACCAGTACAAACTTAATTGTA (SEQ ID NO. 133) 133CCCCGATTTAGAGCTTGACGGGGAAATCAAAA (SEQ ID NO. 134) 134GAATAGCCGCAAGCGGTCCACGCTCCTAATGA (SEQ ID NO. 135) 135GAGTTGCACGAGATAGGGTTGAGTAAGGGAGC (SEQ ID NO. 136) 136GTGAGCTAGTTTCCTGTGTGAAATTTGGGAAG (SEQ ID NO. 137) 137TCATAGCTACTCACATTAATTGCGCCCTGAGA (SEQ ID NO. 138) 138GGCGATCGCACTCCAGCCAGCTTTGCCATCAA (SEQ ID NO. 139) 139GAAGATCGGTGCGGGCCTCTTCGCAATCATGG (SEQ ID NO. 140) 140AAATAATTTTAAATTGTAAACGTTGATATTCA (SEQ ID NO. 141) 141GCAAATATCGCGTCTGGCCTTCCTGGCCTCAG (SEQ ID NO. 142) 142ACCGTTCTAAATGCAATGCCTGAGAGGTGGCA (SEQ ID NO. 143) 143TATATTTTAGCTGATAAATTAATGTTGTATAA (SEQ ID NO. 144) 144TCAATTCTTTTAGTTTGACCATTACCAGACCG (SEQ ID NO. 145) 145CGAGTAGAACTAATAGTAGTAGCAAACCCTCA (SEQ ID NO. 146) 146GAAGCAAAAAAGCGGATTGCATCAGATAAAAA (SEQ ID NO. 147) 147TCAGAAGCCTCCAACAGGTCAGGATCTGCGAA (SEQ ID NO. 148) 148CCAAAATATAATGCAGATACATAAACACCAGA (SEQ ID NO. 149) 149CATTCAACGCGAGAGGCTTTTGCATATTATAG (SEQ ID NO. 150) 150ACGAGTAGTGACAAGAACCGGATATACCAAGC (SEQ ID NO. 151) 151AGTAATCTTAAATTGGGCTTGAGAGAATACCA (SEQ ID NO. 152) 152GCGAAACATGCCACTACGAAGGCATGCGCCGA (SEQ ID NO. 153) 153ATACGTAAAAGTACAACGGAGATTTCATCAAG (SEQ ID NO. 154) 154CAATGACACTCCAAAAGGAGCCTTACAACGCC (SEQ ID NO. 155) 155AAAAAAGGACAACCATCGCCCACGCGGGTAAA (SEQ ID NO. 156) 156TGTAGCATTCCACAGACAGCCCTCATCTCCAA (SEQ ID NO. 157) 157GTAAAGCACTAAATCGGAACCCTAGTTGTTCC (SEQ ID NO. 158) 158AGTTTGGAGCCCTTCACCGCCTGGTTGCGCTC (SEQ ID NO. 159) 159AGCTGATTACAAGAGTCCACTATTGAGGTGCC (SEQ ID NO. 160) 160ACTGCCCGCCGAGCTCGAATTCGTTATTACGC (SEQ ID NO. 161) 161CCCGGGTACTTTCCAGTCGGGAAACGGGCAAC (SEQ ID NO. 162) 162CAGCTGGCGGACGACGACAGTATCGTAGCCAG (SEQ ID NO. 163) 163GTTTGAGGGAAAGGGGGATGTGCTAGAGGATC (SEQ ID NO. 164) 164CTTTCATCCCCAAAAACAGGAAGACCGGAGAG (SEQ ID NO. 165) 165AGAAAAGCAACATTAAATGTGAGCATCTGCCA (SEQ ID NO. 166) 166GGTAGCTAGGATAAAAATTTTTAGTTAACATC (SEQ ID NO. 167) 167CAACGCAATTTTTGAGAGATCTACTGATAATC (SEQ ID NO. 168) 168CAATAAATACAGTTGATTCCCAATTTAGAGAG (SEQ ID NO. 169) 169TCCATATACATACAGGCAAGGCAACTTTATTT (SEQ ID NO. 170) 170TACCTTTAAGGTCTTTACCCTGACAAAGAAGT (SEQ ID NO. 171) 171CAAAAATCATTGCTCCTTTTGATAAGTTTCAT (SEQ ID NO. 172) 172TTTGCCAGATCAGTTGAGATTTAGTGGTTTAA (SEQ ID NO. 173) 173AAAGATTCAGGGGGTAATAGTAAACCATAAAT (SEQ ID NO. 174) 174TTTCAACTATAGGCTGGCTGACCTTGTATCAT (SEQ ID NO. 175) 175CCAGGCGCTTAATCATTGTGAATTACAGGTAG (SEQ ID NO. 176) 176CGCCTGATGGAAGTTTCCATTAAACATAACCG (SEQ ID NO. 177) 177TTTCATGAAAATTGTGTCGAAATCTGTACAGA (SEQ ID NO. 178) 178ATATATTCTTTTTTCACGTTGAAAATAGTTAG (SEQ ID NO. 179) 179AATAATAAGGTCGCTGAGGCTTGCAAAGACTT (SEQ ID NO. 180) 180CGTAACGATCTAAAGTTTTGTCGTGAATTGCG (SEQ ID NO. 181) 181ACCCAAATCAAGTTTTTTGGGGTCAAAGAACG (SEQ ID NO. 182) 182TGGACTCCCTTTTCACCAGTGAGACCTGTCGT (SEQ ID NO. 183) 183TGGTTTTTAACGTCAAAGGGCGAAGAACCATC (SEQ ID NO. 184) 184GCCAGCTGCCTGCAGGTCGACTCTGCAAGGCG (SEQ ID NO. 185) 185CTTGCATGCATTAATGAATCGGCCCGCCAGGG (SEQ ID NO. 186) 186ATTAAGTTCGCATCGTAACCGTGCGAGTAACA (SEQ ID NO. 187) 187TAGATGGGGGGTAACGCCAGGGTTGTGCCAAG (SEQ ID NO. 188) 188ACCCGTCGTCATATGTACCCCGGTAAAGGCTA (SEQ ID NO. 189) 189CATGTCAAGATTCTCCGTGGGAACCGTTGGTG (SEQ ID NO. 190) 190TCAGGTCACTTTTGCGGGAGAAGCAGAATTAG (SEQ ID NO. 191) 191CTGTAATATTGCCTGAGAGTCTGGAAAACTAG (SEQ ID NO. 192) 192CAAAATTAAAGTACGGTGTCTGGAAGAGGTCA (SEQ ID NO. 193) 193TGCAACTAAGCAATAAAGCCTCAGTTATGACC (SEQ ID NO. 194) 194TTTTTGCGCAGAAAACGAGAATGAATGTTTAG (SEQ ID NO. 195) 195AAACAGTTGATGGCTTAGAGCTTATTTAAATA (SEQ ID NO. 196) 196ACTGGATAACGGAACAACATTATTACCTTATG (SEQ ID NO. 197) 197ACGAACTAGCGTCCAATACTGCGGAATGCTTT (SEQ ID NO. 198) 198CGATTTTAGAGGACAGATGAACGGCGCGACCT (SEQ ID NO. 199) 199CTTTGAAAAGAACTGGCTCATTATTTAATAAA (SEQ ID NO. 200) 200GCTCCATGAGAGGCTTTGAGGACTAGGGAGTT (SEQ ID NO. 201) 201ACGGCTACTTACTTAGCCGGAACGCTGACCAA (SEQ ID NO. 202) 202AAAGGCCGAAAGGAACAACTAAAGCTTTCCAG (SEQ ID NO. 203) 203GAGAATAGCTTTTGCGGGATCGTCGGGTAGCA (SEQ ID NO. 204) 204ACGTTAGTAAATGAATTTTCTGTAAGCGGAGT (SEQ ID NO. 205) 205TTTTCGATGGCCCACTACGTAAACCGTC (SEQ ID NO. 206) 206TATCAGGGTTTTCGGTTTGCGTATTGGGAACGCGCG (SEQ ID NO. 207) 207GGGAGAGGTTTTTGTAAAACGACGGCCATTCCCAGT (SEQ ID NO. 208) 208CACGACGTTTTTGTAATGGGATAGGTCAAAACGGCG (SEQ ID NO. 209) 209GATTGACCTTTTGATGAACGGTAATCGTAGCAAACA (SEQ ID NO. 210) 210AGAGAATCTTTTGGTTGTACCAAAAACAAGCATAAA (SEQ ID NO. 211) 211GCTAAATCTTTTCTGTAGCTCAACATGTATTGCTGA (SEQ ID NO. 212) 212ATATAATGTTTTCATTGAATCCCCCTCAAATCGTCA (SEQ ID NO. 213) 213TAAATATTTTTTGGAAGAAAAATCTACGACCAGTCA (SEQ ID NO. 214) 214GGACGTTGTTTTTCATAAGGGAACCGAAAGGCGCAG (SEQ ID NO. 215) 215ACGGTCAATTTTGACAGCATCGGAACGAACCCTCAG (SEQ ID NO. 216) 216CAGCGAAAATTTTACTTTCAACAGTTTCTGGGATTTTGCTAAACTTTT (SEQ ID NO. 217)rt-rem1 AACATCACTTGCCTGAGTAGAAGAACT (SEQ ID NO. 218) rt-rem2TGTAGCAATACTTCTTTGATTAGTAAT (SEQ ID NO. 219) rt-rem3AGTCTGTCCATCACGCAAATTAACCGT (SEQ ID NO. 220) rt-rem4ATAATCAGTGAGGCCACCGAGTAAAAG (SEQ ID NO. 221) rt-rem5ACGCCAGAATCCTGAGAAGTGTTTTT (SEQ ID NO. 222) rt-rem6TTAAAGGGATTTTAGACAGGAACGGT (SEQ ID NO. 223) rt-rem7AGAGCGGGAGCTAAACAGGAGGCCGA (SEQ ID NO. 224) rt-rem8TATAACGTGCTTTCCTCGTTAGAATC (SEQ ID NO. 225) rt-rem9GTACTATGGTTGCTTTGACGAGCACG (SEQ ID NO. 226) rt-rem10GCGCTTAATGCGCCGCTACAGGGCGC (SEQ ID NO. 227)FRET Labeled Staples:

89-TAMRA: (SEQ ID NO: 228) AGAGGCATAATTTCATCTTCTGACTAT/i6-TAMN/AACTA91-Fluorescein: (SEQ ID NO: 229)TATGTAAACCTTT/iFluorT/TTAATGGAAAAATTACCTBiotin Labeled Staples:

77-biotin: (SEQ ID NO. 230)TGCTCAGTCAGTCTCT GAATTTACCAGGAGGT TTTTT /3Bio/ 78-biotin:(SEQ ID NO. 231) GGAAAGCGACCAGGCG GATAAGTGAATAGGTG TTTTT /3Bio/79-biotin: (SEQ ID NO. 232)TGAGGCAGGCGTCAGA CTGTAGCGTAGCAAGG TTTTT /3Bio/ 80-biotin:(SEQ ID NO. 233) TGCCTTTAGTCAGACG ATTGGCCTGCCAGAAT TTTTT /3Bio/

TABLE 3 Staples with probes for TCRα mRNA: A′-73-1GCCACGCTATACGTGG TTTGAAGATATCTTG (SEQ ID NO. 234) A′-73-2GGTGGCGTTGGTCTC CACAGACAACGCTCAT (SEQ ID NO. 235) A′-69-1GCGCAGAGATATCAAA TTTGAAGATATCTTG (SEQ ID NO. 236) A′-69-2GGTGGCGTTGGTCTC ATTATTTGACATTATC (SEQ ID NO. 237) A′-65-1CATATTTAGAAATACC TTTGAAGATATCTTG (SEQ ID NO. 238) A′-65-2GGTGGCGTTGGTCTC GACCGTGTTACCTTTT (SEQ ID NO. 239) A′-61-1TTTTGTTTAAGCCTTA TTTGAAGATATCTTG (SEQ ID NO. 240) A′-61-2GGTGGCGTTGGTCTC AATCAAGAATCGAGAA (SEQ ID NO. 241) A′-57-1AATCACCAAATAGAAA TTTGAAGATATCTTG (SEQ ID NO. 242) A′-57-2GGTGGCGTTGGTCTC ATTCATATATAACGGA (SEQ ID NO. 243) A′-53-1CCTCAAGAATACATGG TTTGAAGATATCTTG (SEQ ID NO. 244) A′-53-2GGTGGCGTTGGTCTC CTTTTGATAGAACCAC (SEQ ID NO. 245)Staples with probes for TCRβ mRNA: B′-158-1AGTTTGGAGCCCTTCA GTGTGACAGGTTTGG (SEQ ID NO. 246) B′-158-2CTGCACTGATGTTCT CCGCCTGGTTGCGCTC (SEQ ID NO. 247) B′-162-1CAGCTGGCGGACGACG GTGTGACAGGTTTGG (SEQ ID NO. 248) B′-162-2CTGCACTGATGTTCT ACAGTATCGTAGCCAG (SEQ ID NO. 249) B′-166-1GGTAGCTAGGATAAAA GTGTGACAGGTTTGG (SEQ ID NO. 250) B′-166-2CTGCACTGATGTTCT ATTTTTAGTTAACATC (SEQ ID NO. 251) B′-170-1TACCTTTAAGGTCTTT GTGTGACAGGTTTGG (SEQ ID NO. 252) B′-170-2CTGCACTGATGTTCT ACCCTGACAAAGAAGT (SEQ ID NO. 253) B′-174-1TTTCAACTATAGGCTG GTGTGACAGGTTTGG (SEQ ID NO. 254) B′-174-2CTGCACTGATGTTCT GCTGACCTTGTATCAT (SEQ ID NO. 255) B′-178-1ATATATTCTTTTTTCA GTGTGACAGGTTTGG (SEQ ID NO. 256) B′-178-2CTGCACTGATGTTCT CGTTGAAAATAGTTAG (SEQ ID NO. 257)

TABLE 4 Primers for reverse transcription linking reaction: CbetaRTACAAGGAGACCTTGGGTGGA (SEQ ID NO. 258) Vbeta1RTPhos′CAGGTGCAGTACAAGGTTCTAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 259) Vbeta2RTPhos′CTGCTGGCACAGAAGTATGTAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 260) Vbeta3RTPhos′GCTAAGCTGCTGGCACAGAAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 261) Vbeta4RTPhos′TCTTAGCTGCTGGCACAGAGAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 262) Vbeta5RTPhos′TCTTGGCTGCTGGCACAAAAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 263) Vbeta12RTPhos′AGAGCTGGCACAGAAGTACAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 264) Vbeta13RTPhos′CATCACTGCTGGCACAGAAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 265) Vbeta14RTPhos′AGAAACTGCTGGCACAGAGAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 266) Vbeta15RTPhos′GCTAAACTGCTGGCACACAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 267) Vbeta16RTPhos′TCTAAGCTGCTTGCACAAAGAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 268) Vbeta17RTPhos′TCTCTACTGCTAGCACAGAGAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 269) Vbeta19RTPhos′CTATACTGCTGGCACAGAGAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 270) Vbeta20RTPhos′TCCCTAGCACCACAGAGATAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 271) Vbeta23RTPhos′GATTGACTGCTGGAGCACAAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 272) Vbeta24RTPhos′TACAGACTGCTGGCACAGAGAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 273) Vbeta26RTPhos′GACAGACTGCTGGCACAGAGAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 274) Vbeta29RTPhos′GCACAGAAGTACACAGATGTAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 275) Vbeta3ORTPhos′TCTCTAGAACTACAGAAATAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 276) Vbeta31RTPhos′AGACTCCAGGCACAGAGGTAAGTGTTCTAGTGTATTCTGTTCCGTCTTTCGTTCTAGCTTGCTGCCTTCTTTTGTCGATAACGTATCGTACCCGTTTAATGGACACTTCCTCATGAGACAGTATCAGAGATCAATTTAGTCCTCAAAGAGTTACTCGTAGTTGCTACGCTCGTTCCGA TGCGAGGATCTTTTAACTGGTA (SEQ ID NO. 277)

TABLE 5 Primers for multiplex PCR reaction: CbetaPCR Phos′GTCACATTTCTCAGATCCTC (SEQ ID NO. 278) Valpha1PCR Phos′TACCTCTGTGCTGTGAGGGA (SEQ ID NO. 279) Valpha2PCR Phos′TTACTGCATTGTGACTGACA (SEQ ID NO. 280) Valpha3PCR Phos′GTACTTCTGCGCAGTCAGTG (SEQ ID NO. 281) Valpha4PCR Phos′CTGGAGGACTCAGGCACTTA (SEQ ID NO. 282) Valpha5PCR Phos′CAGCCTGGAGACTCAGCCAT (SEQ ID NO. 283) Valpha6PCR Phos′GACTCGGCTGTGTACTACTG (SEQ ID NO. 284) Valpha7PCR Phos′GCTCTCTACCTCTGTGCA (SEQ ID NO. 285) Valpha8PCR Phos′GCTGTGTACTTCTGTGCTAC (SEQ ID NO. 286) Valpha9PCR Phos′CTCGGCTGTGTACTTCTGTG (SEQ ID NO. 287) Valpha10PCR Phos′CATCTACTTCTGTGCAGCA (SEQ ID NO. 288) Valpha11PCR Phos′CTACATCTGTGTGGTGGGCG (SEQ ID NO. 289) Valpha12PCR Phos′CAGCTGTCAGACTCTGCCCT (SEQ ID NO. 290) Valpha13PCR Phos′ACAGACTCAGGCACTTAT (SEQ ID NO. 291) Valpha14PCR Phos′TCTCAGCCTGGAGACTCAGC (SEQ ID NO. 292) Valpha15-1PCR Phos′TTCTGTGCTCTCTGGGAGCT (SEQ ID NO. 293) Valpha15-2PCR Phos′TTCTGCGCTCTCTCGGAACT (SEQ ID NO. 294) Valpha16PCR Phos′TATATTTCTGTGCTATG (SEQ ID NO. 295) Valpha17PCR Phos′CAAGTACTTCTGTGCACTGG (SEQ ID NO. 296) Valpha19PCR Phos′TGTACCTCTGCGCAGCAGGT (SEQ ID NO. 297)By way of further example, a detailed outline of a DNA origami method ofmulti-mRNA capture from sorted CD8⁺ T cells is provided as follows inTable 6:

TABLE 6 Day 1 1. Harvest spleen from mouse. a. Add 1 mL RPMI-completemedia to a 1.5 mL tube and go to the mouse house b. Extract spleen frommouse and place into prepared 1.5 mL tube with media, return to lab 2.Digest spleen and lyse RBCs. a. Place a 70 μM cell strainer on one halfof a petri dish b. Pour the spleen/media from the 1.5 mL tube into thestrainer c. Add ~1 mL RPMI-complete media to the strainer using an eyedropper d. Use the base of a plunger from a 3 mL syringe to smash thespleen through the strainer (lift the strainer intermittently to pullthe cells/media through the strainer into the petri dish) e. Rinse theplunger with ~1 mL RPMI-complete media into the strainer and discard theplunger f. Rinse the strainer with ~2 mL RPMI-complete media into thepetri dish, discard the strainer g. Pipet the cells/media from the petridish into a labeled 15 mL tube h. Rinse the petri dish 2X with ~1 mLRPMI-complete media and add to the 15 mL tube i. Centrifuge the tube onProgram 1 (1200 rpm, 5 min, 4 C., A = 9, D = 9, bucket = 3668) j. Pouroff the supernatant and flick the tube to re-suspend the cells k. Add 1mL ACK lysis buffer and incubate 2 min, RT l. Add ~7 mL RPMI-completebuffer (bring total volume to ~8 mL) to quench the lysis buffer m.Centrifuge on Program 1 n. Pour off the supernatant and flick the tubeto re-suspend the cells 3. Sort splenocytes for CD8⁺ T cells a. PrepareMACS buffer (~20 mL/spleen) in a 100 mL glass bottle i. 5 Prepare freshbuffer for experiment ii. In a 100 mL glass bottle add 20 mL autoMACSRinsing Solution iii. Add 1 mL MACS BSA Stock Solution b. Re-suspend thecells with 750 μL MACS buffer c. Add 50 μL MACS CD8a (Ly-2) Microbeadsand mix by pipetting d. Incubate 30 min, in the 4 C. fridge e. Afterincubation add ~5 mL MACS buffer (total volume ~6 mL) f. Centrifuge tubeon Program 1 g. Pour off supernatant and re-suspend the tube by flickingh. Rinse the cells with ~5 mL MACS buffer i. Centrifuge on Program 1 j.Pour off the supernatant and re-suspend the cells by flicking k.Re-suspend the cells in 1 mL MACS buffer l. Set up the MACS columnassembly i. Make sure the magnet is attached to the stand ii. Open a newMACS MS column and place it with the grooves facing outward into one ofthe slots on the magnet (the column should fit snugly into place) iii.If not keeping the non-CD8 cells, place a liquid waste container belowthe column assembly (if keeping the non-CD8 cells place a 15 mL tubebelow the column assembly) m. Place a 70 μL cell strainer upside downover the top of the column n. Prime the column by pipetting 1 mL MACSbuffer onto the cell strainer so that it drips into the column (thebuffer should elute through the column) o. After priming the columnpipet the cells onto the strainer so that they pass through the strainerinto the column p. Rinse the 15 mL tube the cells were in with 1 mL MACSbuffer and pipet this onto the strainer and into the column as well,discard the 15 mL tube q. After the sample has eluted through thecolumn, rinse the column by pipetting 1 mL MACS buffer through thestrainer into the column r. Repeat washing with an additional 1 mL MACSbuffer s. After the column stops dripping label a new 15 mL tube with“Mouse strain, CD8+, Date” t. Elute the CD8⁺ T cells in one quick stepAWAY FROM THE MAGNET! i. Remove the column from the magnet and insert itinto the labeled 15 mL tube ii. Pipet 1 mL MACS buffer directly into thecolumn iii. Use the plunger to slowly elute the cells/media through thecolumn and into the 15 mL tube (press the plunger all the way into thebase of the column) iv. Discard the column/plunger u. Cap the 15 mL tubewith the purified CD8⁺ T cells and place on ice until use. 4.Transfection of purified CD8⁺ T cells with DNA origami a. Obtain a 96well round-bottomed plate and label all wells with corresponding samplenames b. Turn on the ECM 830 BTX electroporator and make sure allsettings are as follows: i. Mode: LV ii. Voltage: 0300 V iii. P. Length:005 ms iv. # Pulses: 01 v. Interval: 200 ms vi. Polarity: UNIPOLAR c.Open a new BTX electroporation cuvette (Blue Cap, 2 mm Gap), and discardthe eye dropper d. Pipet 100 μL cells to the cuvette e. Pipet 25 μLOrigami (50 nM) to the cuvette f. Cap the cuvette and electroporate byplacing the cuvette into the stand with the metal sides of the cuvettefacing the metal terminals of the stand g. Close the cuvette stand andhit “Pulse” on the electroporator h. After electroporating the sample,remove the cap and use a 20 μL pipettor to remove the sample from thecuvette and pipette into the corresponding well of the 96 well plate. i.After removing as much sample as possible from the cuvette, rinse thecuvette with 100 μL Lonza Mouse T cell Nucleofector Media j. Use the 20μL pipettor to remove the media from the cuvette and pipette into thesame well of the 96 well plate (the well should now contain 125 μLcells/origami + 100 μL Nucleofector Media) k. Repeat process for eachsample. Cuvettes can be reused for identical samples, but new cuvettesshould be used for samples receiving different treatments. When finisheddiscard all cuvettes l. Place lid on 96 well plate and incubateovernight (at least 16 hr) in the 37 C./5% CO₂ incubator Day 2 5. Lysecells and purify DNA origami with bound cellular mRNA a. Remove the 96well plate from the incubator b. Centrifuge the plate on Program 4 (1300rpm, 3 min, 4 C., A = 9, D = 9, Bucket = 3670) c. Flick the media fromthe plate d. Re-suspend the cells in 100 μL 1% NP-40 cell lysis buffere. Incubate plate 1 hr, on ice f. Prepare one Sigma Prep Spin Column foreach sample: i. Pipet 50 μL Streptavidin Agarose Resin into a spincolumn ii. Pipet 500 μL 1X TAE-Mg²⁺ into column iii. Centrifuge column10 s, 2000 rpm iv. Remove column from tube and discard effluent v. Capthe bottom of the column with cap provided from Sigma kit and placecolumn back into tube g. Pipet lysate from 96 well plate into column h.Shake tube by hand WITHOUT INVERTING TUBE i. Incubate samples 30 min,RT, shaking every 10 min by hand j. REMOVE CAP FROM COLUMN, and placeinto a PCR rack so you can use later k. Centrifuge column 10 s, 2000 rpml. Wash column 5X: i. Pipet 500 μL 1X TAE Mg²⁺ into column ii.Centrifuge column 10 s, 2000 rpm iii. Discard effluent m. After 5^(th)wash, cap the bottom of the column with the same cap used previously andplace column into a NEW TUBE 6. Reverse transcription a. Reversetranscription will take place directly in the column b. In a PCR tubeprepare the RT master mix using the Qiagen Omniscript RT Kit (note,below recipe is for one sample): 15 μL H₂O  2 μL Buffer  2 μL dNTPs  1μL Ribolock RNase Inhibitor  1 μL CbetaRT primer (100 μM)  3 μL Linkerprimer (10-15 μM)  1 μL Reverse Transcriptase 25 μL Total Volume c. Mixthe master mix by pipetting, and pipet mix directly into the CAPPEDsample column d. Incubate 1 hr, 37 C. heat block (block should be set to~40 C. to account for heat loss through the tube) 7. Ligation a. Removecolumn from heat block b. Ligation will also take place directly in thecolumn c. In a PCR tube prepare the ligation master mix using the NewEngland Biolabs T4 DNA Ligase Kit (note, below recipe is for onesample): 7 μL Buffer 2 μL T4 DNA Ligase 9 μL Total Volume d. Mix mastermix by pipetting, and pipet mix directly into the sample column e.Incubate 1 hr, RT 8. Elution of cDNA a. REMOVE CAP FROM SAMPLE b.Incubate the column 5 min, 95 C. heat block c. Centrifuge column 30 s,2000 rpm (eluted cDNA will be in tube) d. Discard column and keep cDNAon ice until use 9. PCR a. PCR reactions will be performed in standardPCR tubes using Phire Green Hot Start II DNA Polymerase Kit b. Ifrunning multiple samples, prepare one master mix and distribute toindividual PCR tubes and then add cDNA sample to each tube individually(note, below recipe is for one sample)  9.5 μL H₂O   4 μL Buffer   2 μLdNTPs  0.5 μL DMSO 0.75 μL CbetaPCR primer (100 μM) 0.75 μL Valpha PCRprimer (100 μM) 0.70 μL DNA Polymerase 18.2 μL +   2 μL cDNA Sample 20.2μL Total Volume c. Mix PCR sample by pipetting (be sure to remove anyair bubbles) d. Set up the following program on the thermocycler: 98C.-30 s 98 C.-5 s {close oversize brace} 30-40 cycles 45 C.-7 s 72 C.-7s 72 C.-60 s  4 C.-Hold 10. Analyze products by gel electrophoresis(and/or sequencing) a. Prepare a 2% agarose gel while the PCR reactionis running b. Measure 1 g agarose on the scale and add to a 125 mLErlenmeyer flask c. Measure out 50 mL 1× TAE buffer in a 50 mL tube andadd to the flask d. Microwave the flask for 90 s (Be careful, flask willbe extremely hot!) e. Set up gel box/cassette f. Pour the gel from theflask into the cassette, remove any bubbles with a pipette tip g. Insertthe 10 tooth comb with the 1.5 mm width into the grooves on the top ofthe gel cassette (the teeth of the comb should penetrate just below thesurface of the gel) h. Let gel cool/harden for at least 30 min i. Removecomb from gel j. Remove gel cassette from gel box and place the wells onthe negative (black) terminal side k. Fill the gel box with 1× TAE sothat the buffer covers the gel by at least 2-3 cm. l. Prepare PCRsamples (Phire Green Polymerase Kit includes gel loading dye in the PCRbuffer, so no gel loading dye needs to be added): i. Pipet 10 μL 100 bp+DNA loading ladder into a PCR tube ii. To all PCR samples and the ladderadd 1 μL SYBR Gold m. Pipet your ladder into the first well of the geland your PCR samples into the remaining wells (be sure to make a diagramin your lab book of which wells correspond to which samples) n. Run thegel for 1 hr, 110 V o. Remove the gel cassette from the gel box andplace on the UV imager p. Open the program AlphaImager HP q. Hit Acquireand adjust to the following settings: i. Aperture = 1.20 ii. Zoom =25.00 iii. Focus = 1.80 iv. Be sure Auto Expose is NOT checked and setexposure time manually to 0:800 v. Be sure Auto Focus is NOT checked andthat NONE of the display boxes are checked vi. Make sureTransillumination is set to UV vii. Make sure NEITHER of theEPI/Reflective settings are on viii. Make sure the Lens is set to 3(SYBR Green) r. Open the door and adjust the gel placement so that thewells are at the top of the screen and the whole gel is visible s. Closethe door and hit Acquire t. You can adjust the White and Gamma contraststo make the image clearer if necessary u. When finished, click “File →Save Modified → Save Modified Grayscale” v. Save the file into yourfolder as follows “MM.DD.YY Expt Title” w. Discard gel and clean up allwork areas

While the preferred embodiments of the present invention have beenillustrated in detail, it should be apparent that modifications andadaptations to those embodiments may occur to one skilled in the artwithout departing from the scope of the present invention as set forthin the following claims.

What is claimed is:
 1. A method for sequencing genetic information fromindividual cells within a mixed population of cells without single-cellsorting, comprising the steps of: (a) transfecting a cell with a DNAorigami nanostructure; (b) isolating said DNA origami nanostructure fromsaid transfected cell, wherein the DNA origami nanostructure is bound tocomplementary RNA from said cell; (c) reverse transcribing said RNA intocomplementary DNA (cDNA); and (d) sequencing said complementary DNA. 2.A method for obtaining both TCR alpha and beta CDR3 mRNA sequences fromindividual cells within a mixed population of cells without single-cellsorting, comprising the steps of: (a) transfecting a primary T cell witha DNA origami nanostructure having a sequence complementary to TCR alphaand beta constant region mRNA; (b) isolating said DNA origaminanostructure from said transfected cell, wherein the DNA origaminanostructure is bound to said sequences of complementary mRNA from saidcell; (c) reverse transcribing said mRNA into complementary DNA (cDNA);and (d) sequencing said complementary DNA.
 3. The method of claim 2,wherein said DNA origami nanostructures are composed of ssDNA M13 phagerefolded with complementary ssDNA staple sequences into predeterminedshapes with selected staples extended with complementary sequences toTCR alpha and beta constant region mRNA.
 4. The method of claim 1,wherein said step of transfecting comprises electroporation.
 5. A methodfor sequencing two or more nucleic acid sequences from individual cellswithin a mixed population of cells without single-cell sorting,comprising the steps of: (a) transfecting a cell with a DNA origaminanostructure; (b) isolating said DNA origami nanostructure from saidtransfected cell, wherein the DNA origami nanostructure is bound to twoor more sequences of complementary nucleic acids from said cell; and (c)sequencing said two or more sequences of complementary nucleic acids. 6.The method of claim 5, further comprising linking said two or moresequences of complementary nucleic acids into a single DNA molecule andsequencing said DNA.
 7. The method of claim 5, wherein said step oftransfecting comprises electroporation.